POS1038 NAD+ BOOSTERS AS A NOVEL ANTI-INFLAMMATORY TOOL IN RHEUMATOID ARTHRITIS PATIENTS
Authors: C. Perez-Sanchez, A. Escudero Contreras, T. Cerdó, L. Muñoz-Barrera, I. Arias de la Rosa, I. Sanchez-Pareja, L. Marina Mendoza, N. Barbarroja Puerto, M. C. Ábalos-Aguilera, D. Ruiz, J. Moreno, M. I. Burón, J. A. González-Reyes, F. U. Pilar, M. Rojas-Giménez, J. Calvo, E. Collantes Estevez, J. M. Villalba, C. Lopez-Pedrera
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background:</h3> <h3>Objectives</h3> 1- To analyze the levels of NAD<sup>+</sup> and genes involved in its metabolism in Rheumatoid Arthritis (RA) patients. 2- To evaluate the effect of anti-TNF therapy in the NAD<sup>+</sup> metabolism. 3- To explore mechanistically the anti-inflammatory effects of NAD<sup>+</sup> boosters in leukocytes from RA patients. <h3>Methods</h3> Plasma and PBMCs were purified from 100 RA patients and 50 healthy donors (HDs). NAD+ levels were determined by using the NAD/NADH-Glo Assay. Gene expression analysis of markers related to the synthesis (NAMPT, NMNATs, etc), transport (Cx43), and consumption of NAD<sup>+</sup> (PARPs, SIRTs, CD38, etc) were performed in PBMCs by RT-PCR. Public datasets from GEO repository were used to validate the analyses. A panel of 92 inflammatory mediators was also analyzed in the RA serum using Olink platform. In a second cohort of 50 RA patients treated with anti-TNF therapy, NAD+ levels were analyzed before and after 6 months of treatment. PBMCs from a third cohort of 10 active RA patients were treated <i>ex vivo</i> with 1 mM of NAD<sup>+</sup> boosters including nicotinamide (NAM) and nicotinamide riboside (NR) in the presence of inhibitors of NAMPT (FK866) and Cx43 (GAP19). After 24 hours, the Olink inflammatory panel was used to evaluated anti-inflammatory effects. <h3>Results</h3> NAD+ levels were significantly reduced in the plasma of RA patients and the expression of genes involved in the consumption (PARPs, SIRTs, CD38, CD157) and biosynthesis (NMNATs) of NAD<sup>+</sup> were increased and reduced respectively in RA PBMCs. NAMPT and Cx43 were also upregulated in those patients. The results were validated in an independent dataset. Unsupervised clustering analysis of the inflammatory proteome identified 3 cluster of patients, where cluster 1 was characterised by higher levels of inflammatory mediators and disease activity (DAS28) and lower levels of NAD<sup>+</sup>, and cluster 3 by lower levels of inflammatory mediators and DAS28 and higher levels of NAD<sup>+</sup>. Cluster 2 showed intermediate profiles. Anti-TNF therapy restored the altered NAD<sup>+</sup> levels in RA patients towards those exhibited by HD. Moreover, changes in DAS28 and NAD<sup>+</sup> levels were correlated. In vitro, NAD<sup>+</sup> boosters promoted a significant increase in NAD<sup>+</sup> levels of RA PBMCs. The FK866 treatment highlighted the key role of NAMPT in the generation of NAD<sup>+</sup> promoted by NAM. The inhibition of the extracellular transport of NAD<sup>+</sup> with GAP19 further increased the intracellular NAD<sup>+</sup> content in the presence of NAM and NR. Both boosters reduced the levels of numerous secreted inflammatory mediators in the supernatant, which was enhanced by effect of GAP19. In parallel, the gene expression of key inflammatory mediators was deeply reduced along with markers of oxidative stress and apoptosis. <h3>Conclusion</h3> 1- The NAD<sup>+</sup> levels and its metabolism is altered in RA patients and directly linked to their inflammatory profile and disease activity. 2- Anti-TNF therapy restore NAD+ altered levels in line with the clinical response. 3- NR and NAM increase the NAD<sup>+</sup> levels in PBMCs from active RA patients, through a mechanism mediated in part by NAMPT, promoting an extensive anti-inflammatory effect which is enhanced by Cx43 inhibition. NAD<sup>+</sup> boosters might be considered as novel therapeutic approach to reduce inflammation in RA. Supported by: RYC2021-033828-I, MCIN/AEI/10.13039/501100011033 and "NextGenerationEU"/PRTR. CLP lab: ISCIII (PI21/0591, CD21/00187 and RICORS, RD21/0002/0033), JdA (P20_01367) co-financed by FEDER and FAR. JMV lab: MCIU/AEI grants RTI2018-100695-B-I00, PID2021-126280OB-I00 and PRE2019-087438 (FPI); JdA (P18-RT-4264, 1263735-R and BIO-276), (FEDER); and UCO (PPIT-2022E-026295). <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.